A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

Abstract

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

Identification of genes required for long term survival of L. pneumophila in water

“…We next directed our analyses to individual members of the candidate list of water stress survival genes, focusing on proteins not previously noted as important for survival in water starvation conditions (Table 1), to validate the predictions made by our Tn-seq analysis. For this purpose, we took advantage of the recently established CRISPR interference system for L. pneumophila (27). This system utilizes an anhydrotetracycline (ATC)-inducible dcas9 gene inserted in the nonfunctional chromosomal thyA locus of L pneumophila strain Lp02, and crRNA-as well as tracrRNA-encoding sequences on a plasmid (27, 28).…”

“…Final plasmids were constructed by introducing pMME985 (tracrRNA) and a preliminary plasmid into pMME977 by the Gateway LR reaction. After confirmation of single clones, the final plasmids were electroporated into L. pneumophila Philadelphia-1 Lp02dCas9Lux and selected on plates without thymidine (27).…”

“…Previous work has demonstrated that CRISPRi technology allows efficient gene knockdowns in L. pneumophila during growth in either broth or phagocytic cells (27). Its applicability to prolonged water starvation conditions has not been previously established.…”

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

“…Additionally, CRISPRi/a can be used to control transcription regulatory networks, such as genetic circuits, by designing and expressing gRNA to regulate the output promoter for each logic gate or node ( Figure 1B ). CRISPRi/a is especially effective for controlling complex synthetic transcription regulatory networks as the gRNA can be designed to target nearly any arbitrary sequence with an appropriate PAM (or equivalent) sequence ( Taketani et al, 2020 ; Ellis et al, 2021 ). CRISPRi/a circuits can be fully synthetic and auxiliary to the native genetic regulatory networks, such as a heterologous sensor or multi-input circuit that senses and responds to external inputs in complex environments ( Mimee et al, 2015 ; Taketani et al, 2020 ).…”

“…CRISPRi is particularly useful for investigating essential genes because its repression can be titrated to prevent full knockdown and cell death ( Knoot et al, 2020 ; Bosch et al, 2021 ). Additionally, epistatic effects of multiple genes can easily be investigation by simply expressing multiple gRNA within the same cell ( Ellis et al, 2021 ; McNeil et al, 2022 ). Although not as common as CRISPRi due to stricter design rules ( Fontana et al, 2020a ), CRISPRa can be used to induce expression of silent genes to investigate their functions and products, including entire silent biosynthetic gene clusters ( Ke et al, 2021 ).…”